Name: a method to assess fc-mediated effector functions induced by influenza hemagglutinin specific antibodies
Uploaded: 2018-02-23T12:30:00
Description: Watch this Scientific Journal Video about A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies at JoVE.com

Dette prosjektet har blitt finansiert delvis med føderale midler fra National Institute of Allergy og smittsomme sykdommer, National Institutes of Health, Department of Health and Human Services, under CEIRS kontrakt P01AI097092-04S1 (P.E.L.).

Eksperimenter utført i dette manuskriptet ble gjort følgende Icahn School of Medicines institusjonelle koden for etikk og forskrifter.
1. uttrykk for influensa Virus Hemagglutinin via Transfection eller infeksjon
Hva:
<ol><li>Plate menneskelige embryonale (HEK 293T) nyreceller på en tetthet av 2 x 104 celler/godt i en hvit vev kultur behandlet 96-brønns plate og la det sitte 4 h i en 37 °C inkubator (med 5% CO...

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	<li>Hobson, D., Curry, R. L., Beare, A. S., Ward-Gardner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+serum+haemagglutination-inhibiting+antibody+in+protection+against+challenge+infection+with+influenza+A2+and+B+viruses.">The role of serum haemagglutination-inhibiting antibody in protection against challenge infection with influenza A2 and B viruses.</a> J Hygiene. 70 (04), 767-777 (1972).</li><li>Throsby, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterosubtypic+neutralizing+monoclonal+antibodies+cross-protective+against+H5N1+and+H1N1+recovered+from+human+IgM++memory+B+cells.">Heterosubtypic neutralizing monoclonal antibodies cross-protective against H5N1 and H1N1 recovered from human IgM+ memory B cells.</a> PloS One. 3 (12), e3942 (2008).</li><li>Ekiert, D. 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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+common+solution+to+group+2+influenza+virus+neutralization.">A common solution to group 2 influenza virus neutralization.</a> PNAS. 111 (1), 445-450 (2014).</li><li>Dreyfus, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+Conserved+Protective+Epitopes+on+Influenza+B+Viruses.">Highly Conserved Protective Epitopes on Influenza B Viruses.</a> Science. 337 (6100), 1343-1348 (2012).</li><li>Krammer, F., Palese, P., Steel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+Universal+Influenza+Virus+Vaccine+Design+and+Antibody+Mediated+Therapies+Based+on+Conserved+Regions+of+the+Hemagglutinin.">Advances in Universal Influenza Virus Vaccine Design and Antibody Mediated Therapies Based on Conserved Regions of the Hemagglutinin.</a> Curr Top Microb and Immunol. , (2014).</li><li>Impagliazzo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+stable+trimeric+influenza+hemagglutinin+stem+as+a+broadly+protective+immunogen.">A stable trimeric influenza hemagglutinin stem as a broadly protective immunogen.</a> Science. , (2015).</li><li>Nachbagauer, R., Krammer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+Microbiology+and+Infection.">Clinical Microbiology and Infection.</a> Clin Microb Infect. , 1-7 (2017).</li><li>Krammer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+universal+influenza+virus+vaccine+approaches.">Novel universal influenza virus vaccine approaches.</a> Current opinion in virology. 17, 95 (2016).</li><li>Steel, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+virus+vaccine+based+on+the+conserved+hemagglutinin+stalk+domain.">Influenza virus vaccine based on the conserved hemagglutinin stalk domain.</a> mBio. 1 (1), (2010).</li><li>Krammer, F., Pica, N., Hai, R., Margine, I., Palese, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+hemagglutinin+influenza+virus+vaccine+constructs+elicit+broadly+protective+stalk-specific+antibodies.">Chimeric hemagglutinin influenza virus vaccine constructs elicit broadly protective stalk-specific antibodies.</a> J Virol. 87 (12), 6542-6550 (2013).</li><li>Krammer, F., Pica, N., Hai, R., Tan, G. S., Palese, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemagglutinin+Stalk-Reactive+Antibodies+Are+Boosted+following+Sequential+Infection+with+Seasonal+and+Pandemic+H1N1+Influenza+Virus+in+Mice.">Hemagglutinin Stalk-Reactive Antibodies Are Boosted following Sequential Infection with Seasonal and Pandemic H1N1 Influenza Virus in Mice.</a> J Virol. 86 (19), 10302-10307 (2012).</li><li>Hai, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+viruses+expressing+chimeric+hemagglutinins:+globular+head+and+stalk+domains+derived+from+different+subtypes.">Influenza viruses expressing chimeric hemagglutinins: globular head and stalk domains derived from different subtypes.</a> J Virol. 86 (10), 5774-5781 (2012).</li><li>Dunand, C. J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Both+Neutralizing+and+Non-Neutralizing+Human+H7N9+Influenza+Vaccine-Induced+Monoclonal+Antibodies+Confer+Protection.">Both Neutralizing and Non-Neutralizing Human H7N9 Influenza Vaccine-Induced Monoclonal Antibodies Confer Protection.</a> Cell Host and Microbe. 19 (6), 800-813 (2016).</li><li>Tan, G. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broadly-Reactive+Neutralizing+and+Non-neutralizing+Antibodies+Directed+against+the+H7+Influenza+Virus+Hemagglutinin+Reveal+Divergent+Mechanisms+of+Protection.">Broadly-Reactive Neutralizing and Non-neutralizing Antibodies Directed against the H7 Influenza Virus Hemagglutinin Reveal Divergent Mechanisms of Protection.</a> PLoS Path. 12 (4), e1005578 (2016).</li><li>DiLillo, D. J., Tan, G. S., Palese, P., Ravetch, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broadly+neutralizing+hemagglutinin+stalk-specific+antibodies+require+Fc&#947;R+interactions+for+protection+against+influenza+virus+in+vivo.">Broadly neutralizing hemagglutinin stalk-specific antibodies require Fc&#947;R interactions for protection against influenza virus in vivo.</a> Nat Med. 20 (2), 143-151 (2014).</li><li>DiLillo, D. J., Palese, P., Wilson, P. C., Ravetch, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broadly+neutralizing+anti-influenza+antibodies+require+Fc+receptor+engagement+for+in+vivo+protection.">Broadly neutralizing anti-influenza antibodies require Fc receptor engagement for in vivo protection.</a> J Clin Invest. 126 (2), 605-610 (2016).</li><li>Potter, C. W., Oxford, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinants+of+immunity+to+influenza+infection+in+man.">Determinants of immunity to influenza infection in man.</a> Brit Med Bull. 35 (1), 69-75 (1979).</li><li>Memoli, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+Antihemagglutinin+and+Antineuraminidase+Antibodies+as+Correlates+of+Protection+in+an+Influenza+A/H1N1+Virus+Healthy+Human+Challenge+Model.">Evaluation of Antihemagglutinin and Antineuraminidase Antibodies as Correlates of Protection in an Influenza A/H1N1 Virus Healthy Human Challenge Model.</a> mBio. 7 (2), e00417 (2016).</li><li>Jegaskanda, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-associated+cross-reactive+antibody-dependent+cellular+cytotoxicity+toward+2009+pandemic+influenza+A+virus+subtype+H1N1.">Age-associated cross-reactive antibody-dependent cellular cytotoxicity toward 2009 pandemic influenza A virus subtype H1N1.</a> J Infect Dis. 208 (7), 1051-1061 (2013).</li><li>Jegaskanda, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-Reactive+Influenza-Specific+Antibody-Dependent+Cellular+Cytotoxicity+Antibodies+in+the+Absence+of+Neutralizing+Antibodies.">Cross-Reactive Influenza-Specific Antibody-Dependent Cellular Cytotoxicity Antibodies in the Absence of Neutralizing Antibodies.</a> J Immunol. 190 (4), 1837-1848 (2013).</li><li>Jegaskanda, S., Wheatley, A. K., Kent, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ScienceDirectAntibody-dependent+cellular+cytotoxicity+and+influenza+virus.">ScienceDirectAntibody-dependent cellular cytotoxicity and influenza virus.</a> Curr Opin Virol. 22 (C), 89-96 (2017).</li><li>Srivastava, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Dominant+Antibody-Dependent+Cell-Mediated+Cytotoxicity+Epitopes+on+the+Hemagglutinin+Antigen+of+Pandemic+H1N1+Influenza+Virus.">Identification of Dominant Antibody-Dependent Cell-Mediated Cytotoxicity Epitopes on the Hemagglutinin Antigen of Pandemic H1N1 Influenza Virus.</a> J Virol. 87 (10), 5831-5840 (2013).</li><li>He, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epitope+specificity+plays+a+critical+role+in+regulating+antibody-dependent+cell-mediated+cytotoxicity+against+influenza+A+virus.">Epitope specificity plays a critical role in regulating antibody-dependent cell-mediated cytotoxicity against influenza A virus.</a> PNAS. 113 (42), 11931-11936 (2016).</li><li>Leon, P. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+activation+of+Fc-mediated+effector+functions+by+influenza+virus+hemagglutinin+antibodies+requires+two+points+of+contact.">Optimal activation of Fc-mediated effector functions by influenza virus hemagglutinin antibodies requires two points of contact.</a> PNAS. , 201613225 (2016).</li><li>Benjamin, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Broadly+Neutralizing+Human+Monoclonal+Antibody+Directed+against+a+Novel+Conserved+Epitope+on+the+Influenza+Virus+H3+Hemagglutinin+Globular+Head.">A Broadly Neutralizing Human Monoclonal Antibody Directed against a Novel Conserved Epitope on the Influenza Virus H3 Hemagglutinin Globular Head.</a> J Virol. 88 (12), 6743-6750 (2014).</li><li>Ekiert, D. C., Kashyap, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-neutralization+of+influenza+A+viruses+mediated+by+a+single+antibody+loop.">Cross-neutralization of influenza A viruses mediated by a single antibody loop.</a> Nature. 488 (7417), 526-532 (2013).</li><li>He, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broadly+Neutralizing+Anti-Influenza+Virus+Antibodies:+Enhancement+of+Neutralizing+Potency+in+Polyclonal+Mixtures+and+IgA+Backbones.">Broadly Neutralizing Anti-Influenza Virus Antibodies: Enhancement of Neutralizing Potency in Polyclonal Mixtures and IgA Backbones.</a> J Virol. 89 (7), 3610 (2015).</li><li>Kristensen, A. 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Requirement for antibody and non-T lymphocytes.</a> J Immunol (Baltimore, Md. : 1950). 119 (6), 2100-2106 (1977).</li><li>Corrales-Aguilar, E., Trilling, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+assay+for+detecting+virus-specific+antibodies+triggering+activation+of+Fc&#947;+receptors.">A novel assay for detecting virus-specific antibodies triggering activation of Fc&#947; receptors.</a> J Immunol Meth. 387 (1-2), 21-35 (2013).</li><li>de Vries, R. 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Metoden beskrevet her tillater brukeren å måle hvor en HA-spesifikke monoklonale antistoffer til å engasjere seg i murine FcγRIV. Målet antigen, influensa virus hemagglutinin, er uttrykt på overflaten av cellene etter infeksjon av virus eller hva av plasmider DNA. Analysen er flere mottagelig å bruke ulike overflaten-uttrykt virale proteiner sammen med andre FcγRs (mennesker eller Trouble). I tillegg, vi har brukt sera prøver for å vurdere muligheten av antistoffer i en polyklonale sammenheng å overtale Fc fun...

Immunitet fra sesongens influensavaksine har tradisjonelt blitt vurdert av hemagglutination hemming (HI) analysen1, som måler tilstedeværelse av antistoffer som mål reseptor binding området av hemagglutinin. Disse HI-aktiv antistoffer kan tildele sterilisering immunitet, men er generelt smale i deres bredden i beskyttelse, gi immunitet til bare en velge noen stammer av influensavirus. Isolering og karakterisering av grovt reaktive monoklonale antistoffer som gjenkjenner hemagglutinin (HA) tyder utviklingen av en universal influensavaksine er innen rekkevidde2,3,4, 5 , 6 , 7 , 8. en av de viktigste målene for en universell influensavaksine er å indusere en sterk antistoffet svaret mot de konserverte områdene av influensa virus9,10,11,12 , 13 , 14 , 15 , 16. bredt reaktive antistoffer som gjenkjenner disse bevarte epitopes, består av både nøytraliserende og ikke-nøytralisere antistoffer17,18, har vist seg å kreve Fc-FcγR vekselsvirkningene for optimal beskyttelse i vivo og høydepunkt bidrag av Fc-mediert immunitet til generelle immunforsvaret til influensa virus19,20.
Korrelerer beskyttelse er avgjørende i å vurdere immunitet mot infeksjon. Disse verdiene kan forskere og klinikere å beregne effekten av vaksiner, betydningen av data, og i løpet av behandling. Den eneste etablerte relateres til beskyttelse mot influensa virus smitte er hemagglutination hemming analysen. En titer 1:40 er forbundet med en 50% reduksjon i risikoen for sykdom1,21 - og gjenspeiler tilstedeværelse av antistoffer som hemmer agglutinerende ved målretting reseptoren bindende område ligger på globular hodet av HA. Men bør det også bemerkes at T-celle svar kan være en bedre relateres til beskyttelse mot influensa virusinfeksjon hos eldre. Interessant antyder en fersk rapport at neuraminidase hemming aktivitet kanskje være en bedre prediktor for immunitet til influensa22. Heldigvis, typisk i vitro søk som nøytralisering eller neuraminidase hemming analyser kan måle HA stengel - eller neuraminidase-spesifikke antistoffer. De fleste av disse konvensjonelle i vitro analyser, men bare tar hensyn til funksjonen i antigen bindende regionen antistoffer og måler ikke rollen regionen Fc. I tillegg bidrag av ikke-nøytralisere Antistoffene det beskytte via Fc reseptor engasjement i vivo er ikke oppdaget17,18. For å måle vern av antistoffer som induserer Fc-mediert immunitet som antistoff avhengige celle mediert cytotoksisitet (ADCC), er en robust i vitro analysen nødvendig.
Metoden beskrevet nedenfor vurderer muligheten for murine monoklonale antistoffer å overtale Fc-mediert funksjoner ved hjelp av en genetisk modifisert Jurkat celle linje uttrykke aktiverings murint type 1 FcR, FcγRIV. Antistoff engasjement i FcγR transduces intracellulær signaliserer at utløsere kjernefysiske faktor aktivert T-celler-mediert luciferase aktivitet. Analysen har flere fordeler sammenlignet med tradisjonelle teknikker som krever bruk av flowcytometri å merker aktivisering Fc-mediert effektor funksjoner19,23,24, isolasjon og kultur av primære Effektor celler ,25,26. Først kan protokollen beskrevet her lett tilpasses å inkorporere ulike viral mål, FcγRs og antistoffer fra ulike arter (menneskelige og murine). Andre, bruk av en modifisert Jurkat celle linje uttrykke en FcγR med en luciferase reporter gene under en kjernefysiske faktor på aktivert T-celle (NFAT) gir en stort format analysen som kan analyseres lett bruke en plate leser måle luminescence. Her, erstattes tradisjonelle aktivering av primære Effektor celler med induksjon av NFAT-luciferase på antistoff engasjement i en FcγR på overflaten av Jurkat cellen. Denne 96-brønns plate format analysen kan for måling av opptil fire forskjellige prøver triplicates med syv fortynninger-en rekke prøver som kan være tungvint benytter tradisjonell teknikker. Det er flere punkter du bør vurdere med denne analysen som kan påvirke utformingen av eksperimenter og tolkning av data. Viral målet må uttrykkes på cellens overflate for at antistoff anerkjennelse. For å løse dette, kan målet antigen (f.eks en intern viral protein) være direkte belagt på overflaten av en plate. Men har dette ennå ikke blitt grundig testet. I tillegg den endrede Jurkat celle linjen uttrykker bare én type aktivere FcγR og uttrykker ikke noen hemmende FcγRs, mens primære cellelinjer uttrykke alle reseptorer i fysiologiske sammenheng.
Vi har brukt denne analysen til å demonstrere at epitope spesifisitet i en polyklonale sammenheng spiller en avgjørende rolle i å regulere Fc-mediert effektor funksjoner og at optimal aktivering av antistoff-avhengig celle-mediert respons krever to punkter av Kontakt27,28. Her beskriver vi en metode som vurderer evne til strå-spesifikke monoklonale antistoffer å engasjere og aktivere murint aktivere FcγRIV. Men det er unntak29,30, et stort antall bredt reaktive antistoffer målrette antigenically bevart stilken regionen i hemagglutinin og dermed spille en viktig rolle i utviklingen av en universal influensa vaksine.

<table border="0" cellpadding="0" cellspacing="0" width="921">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">A549 cells&#160;</td>
			<td style="width:141px;">ATCC</td>
			<td style="width:184px;">CCL-185</td>
			<td style="width:353px;">Adenocarcinomic human alveolar basal epithelial cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">HEK 293T cells</td>
			<td style="width:141px;">ATCC</td>
			<td style="width:184px;">CRL-3216</td>
			<td>Human embryonic kidney cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Lipofectamine 2000</td>
			<td style="width:141px;">Life Technologies&#160;</td>
			<td style="width:184px;">11668019</td>
			<td>Transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1X Opti-MEM Reduced Serum Medium</td>
			<td>ThermoFisher Scientific</td>
			<td style="width:184px;">51985-034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">White tissue culture treated 96-well plate</td>
			<td>Corning, Inc.&#160;</td>
			<td>3917</td>
			<td>Assay plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">10X MEM</td>
			<td style="width:141px;">Gibco</td>
			<td style="width:184px;">11430-030</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Jurkat cell expressing murine FcgRIV</td>
			<td style="width:141px;">Promega</td>
			<td style="width:184px;">M1201</td>
			<td>Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Jurkat cell expressing human FcgRIIIa</td>
			<td style="width:141px;">Promega</td>
			<td style="width:184px;">G7010</td>
			<td>Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Jurkat cell expressing human FcgRIIa</td>
			<td style="width:141px;">Promega</td>
			<td style="width:184px;">G9901</td>
			<td>Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luminometer</td>
			<td>BioTek</td>
			<td>Synergy H1 Multi-Mode reader</td>
			<td>Luminescence plate reader</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Bio-Glo Luciferase Assay System</td>
			<td style="width:141px;">Promega</td>
			<td style="width:184px;">G7940</td>
			<td>Contains luciferase assay buffer and luciferase assay substrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Madin Darby canine kidney cells</td>
			<td style="width:141px;">ATCC</td>
			<td style="width:184px;">CCL-34</td>
			<td>Canine kidney cells</td>
		</tr>
	</tbody>
</table>

a method to assess fc-mediated effector functions induced by influenza hemagglutinin specific antibodies

Antistoffer spille en avgjørende rolle i koblingen immunreaksjoner medfødte og adaptive mot viral patogener gjennom antigen bindende domener og Fc-regioner. Her beskriver vi hvordan måle aktiveringen av Fc funksjoner effektor av monoklonale antistoffer målretting influensa virus hemagglutinin med bruk av en genmodifisert Jurkat celle linje uttrykke aktiverings type 1 Fc-FcγR. med denne metoden, den bidrag av bestemte Fc-FcγR vekselsvirkningene av immunglobuliner kan bestemmes i vitro analysen.

Vi viste at en stengel-spesifikke mAb, 6F12, men ikke hodet-spesifikke en hode-spesifikke mAb, PY102, kunne aktivere primære naturlige drepe celler av upregulating CD107a og interferon γ19. For å modellere evne til en stilk-spesifikke antistoffer å aktivere primære menneskelige NK celler, viser vi at en stengel-spesifikke mAb, 6F12, robust kan aktivere modifisert Jurkat cellen uttrykke murine FcγRIV av ~ 14-fold. På den annen side, gjør leder-spesifikke mAb, PY102 og kontrollen IgG ikke (<...

Watch this Scientific Journal Video about A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies at JoVE.com

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Vi beskriver en metode for å måle aktivering av funksjoner Fc-mediert effektor av antistoffer som er rettet mot influensa virus hemagglutinin. Denne analysen kan også tilpasses for å vurdere muligheten av monoklonale antistoffer eller polyklonale sera målretting andre viral overflate glykoproteiner å overtale Fc-mediert immunitet.

Metode for å vurdere funksjoner Fc-mediert effektor av influensa Hemagglutinin spesifikke antistoffer

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

The overall goal of this protocol is to assess the ability of an antibody or a serum sample to activate Fc-mediated effector function. This method can help answer key questions in the immunology and vaccinology fields by measuring the activation of antibody-dependent Fc-mediated immunity. The main advantage of this technique is that the experiment can be done within 24 hours from the material preparation to the data analysis.Demonstrating this procedure will be Mark Bailey, graduate student from the laboratory of Peter Palese. To induce influenza virus hemagglutinin expression via transfection, first plate human embryonic kidney 293 cells at a two times 10 to the fourth cells per well concentration in a white tissue culture treated 96-well plate. After four hours at 37 degrees Celsius and 5%CO2, transfect the cells with 100 nanograms of DNA coding for viral hemagglutinin and 0.2 microliters of transfection reagent per well and a total volume of 50 microliters of 1X optimum-reduced serum medium.Add DNA liposome complexes to each well to transfect and return the plate to the incubator for another 18 hours. To induce influenza virus hemagglutinin expression via infection, plate A549 cells at a two times 10 to the fourth cells per well concentration in a white tissue culture treated 96-well plate for an at least 18-hour incubation at 37 degrees Celsius and 5%CO2. At the end of the incubation, dilute the influenza virus to a multiplicity infection of three in 100 microliters of 1X MEM medium per well and infect the A459 cells for one hour in the cell culture incubator.At the end of the incubation, replace the inoculum with 100 microliters of fresh 1X MEM medium without virus and return the cells to the incubator for another 18 to 24 hours. To assess the ability of specific antibodies of interest to target the hemagglutinin-expressing cells, replace the supernatant of the virally-affected cell cultures with 25 microliters of Antibody-Dependent Cell-mediated Cytotoxicity or ADCC assay medium. Next, in a separate 96-well plate, perform four full dilutions of the antibodies of interest in triplicate in fresh ADCC medium starting at 10 micrograms per milliliter according to the schematic and taking care to include the background and no antibody controls.When all of the antibody has been diluted, transfer 25 microliters of the diluted antibody to the appropriate corresponding wells in the experimental cell plate and place the plate in the cell culture incubator. After 15 minutes, add 7.5 times 10 to the fourth modified effector Jurkat cells expressing the appropriate Fc receptor per well in 25 microliters of ADCC medium for a final volume of 75 microliters of ADCC medium per well. Return the plate to the cell culture incubator for six hours thawing luciferase substrate buffer in a 37 degree Celsius water bath during the last hour of the incubation.When the buffer is ready, add 75 microliters of luciferase substrate buffer to all of the wells except the background control well and read the plate on a luminometer. The fold induction of antibody-dependent cell-mediated cytotoxicity in the transfected or infected cell cultures can then be calculated. In this experiment, a stock-specific monoclonal antibody robustly activated and modified Jurkat cells expressing murine Fc gamma receptor four by approximately 14-fold compared to head-specific monoclonal and control IgG antibodies.After watching this video, you should have a good understanding of how to set up an experiment for assessing the ability of specific antibodies of interest to activate Fc-mediated immunity.

Research

JoVE Journal

Immunology and Infection

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

High-throughput Detection Method for Influenza Virus

High-throughput Detection Method for influensavirus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

In Vitro analysen å evaluere virkningen av immunoregulatory Pathways på HIV-spesifikke CD4 T Cell effektorfunksjon

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Introduksjon Kokulturanalyse for å vurdere patogenindusert nøytrofil transepiteriemigrasjon

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

Expression of Functional rekombinant hemagglutinin og neuraminidase Proteiner fra Novel H7N9 Influensa virus Bruke Baculovirus Expression System

The baculovirus expression system is a powerful tool for expression of  recombinant proteins. Here we use it to produce correctly folded and  glycosylated ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

In Vitro Demontering av influensa A virus kapsider ved gradientsentrifugering

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Bruk av Fluorescence Resonance Energy Transfer (FRET) for å undersøke Effector Trans Effektivitet av<em&gt; Coxiella burnetii</em&gt; Under siRNA lyddemping

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

Fluorescens-baserte Neuraminidase inhiberingsanalyse for å vurdere i hvilken grad influensavirus til neuraminidase Inhibitor Class of Antivirale

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Generasjon av Escape varianter av nøytralisere influensa Virus monoklonale antistoffer

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Måle influensa nøytralisere antistoff Svar å A(H3N2) virus i menneskelig Sera av Microneutralization søk ved hjelp av MDCK-SIAT1 celler

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...